Fibrosis, the formation of excessive amounts of fibrotic or scar tissue, is a central issue in medicine. Scar tissue blocks arteries, immobilizes joints and damages internal organs, wreaking havoc on the body""s ability to maintain vital functions. Every year, about 1.3 million people are hospitalized due to the damaging effects of fibrosis, yet doctors have few therapeutics to help them control this dangerous condition. As a result, they often see patients crippled, disfigured or killed by unwanted masses of uncontrollable scars.
Fibrosis can follow surgery in the form of adhesions, keloid tumors or hypertrophic (very severe) scarring. Fibrosis causes contractures and joint dislocation following severe bums, wounds or orthopaedic injuries; it can occur in any organ and accompanies many disease states, such as hepatitis (liver cirrhosis), hypertension (heart failure), tuberculosis (pulmonary fibrosis), scleroderma (fibrotic skin and internal organs), diabetes (nephropathy) and atherosclerosis (fibrotic blood vessels).
Ironically, the very process designed to repair the body can lead to dangerous complications. Like epoxy, scar tissue serves only a structural role. It fills in the gaps, but cannot contribute to the function of the organ in which it appears. For example, as fibrotic scar tissue replaces heart muscle damaged by hypertension, the heart becomes less elastic and thus less able to do its job. Similarly, pulmonary fibrosis causes the lungs to stiffen and decrease in size, a condition that can become life-threatening. Fibrotic growth can also proliferate and invade the healthy tissue that surrounds it even after the original injury heals. Too much scar tissue thus causes physiological roadblocks that disfigure, cripple or kill.
In most cases, fibrosis is a reactive process, and several different factors can apparently modulate the pathways leading to tissue fibrosis. Such factors include the early inflammatory responses, local increase in fibroblast cell populations, modulation of the synthetic function of fibroblasts, and altered regulation of the biosynthesis and degradation of collagen.
One treatment approach, therefore, has been to target the early inflammatory response. Treatment with topical corticosteroids has achieved limited success, if used early in fibrosis. However, steroid therapy has little or no effect once scar tissue has already formed. Furthermore, prolonged administration of hydrocortisone, in pulmonary fibrotic disease for example, may actually worsen the condition.
The second approach involves slowing the proliferation of those cells responsible for the increased collagen synthesis. Generally, this involves fibroblast cells, except in the vasculature where smooth muscle cells are responsible for collagen deposition. Compounds that have been used to inhibit fibroblast proliferation include benzoic hydrazide, as taught by U.S. Pat. No. 5,374,660. Benzoic hydrazide has been shown to suppress collagen synthesis and fibroblast proliferation, at least in tissue culture cells. U.S. Pat. No. 5,358,959 teaches the use of imidazole derivatives to inhibit the growth of fibroblasts by blocking the calcium-activated potassium channel. This particular agent also inhibits the proliferation of endothelial cells and vascular smooth muscle cells.
Likewise, a number of agents which affect smooth muscle cell proliferation have been tested. These compositions have included heparin, coumarin, aspirin, fish oils, calcium antagonists, steroids, prostacyclin, rapamycin, dipyridamole, ultraviolet irradiation, gamma (.gamma.)-interferon, serotonin inhibitors, methotrexate and mycophenolic acid, either alone or in various combinations.
A number of treatments have been devised that are based on the modulation of the synthetic function of fibroblast or smooth muscle cells. Like most cells, fibroblasts and smooth muscles cells are modulated by cytokines (factors secreted in response to infection that modify the function of target cells). Gamma interferon is a lymphokine (a cytokine that is produced by lymphocytes) known to inhibit fibroblast proliferation and collagen synthesis. Likewise, the monokine (a cytokine that is produced by macrophages) beta-interferon serves the same function. Thus, U.S. Pat. No. 5,312,621 teaches the use of these cytokines in the treatment of fibrosis. Similarly, certain cytokines have been tested for their effect on the proliferation and stimulation of collagen synthesis in smooth muscle cells. For example, U.S. Pat. No. 5,268,358 is directed to the use of peptides that block the binding of platelet-derived growth factors to their receptors. U.S. Pat. No. 5,304,541 is directed to chimeric transforming growth factor-beta (TGF-.beta.) peptides which block cell proliferation. U.S. Pat. No. 5,308,622 is directed to conjugates comprising fibroblastic growth factor (FGF) and cytotoxic agents. U.S. Pat. No. 5,326,559 is directed to interleukin-2 targeted molecules. Although promising, many of these agents and compositions have known and serious side effects and, consequently, limited effectiveness.
The final treatment strategy involves directly influencing the metabolism of collagen and the other components of fibrotic tissue. Thus, drugs that interfere with the biosynthesis, accumulation and catabolism of collagen have been used in the treatment of fibrosis. Many drugs are used to inhibit collagen synthesis, including derivatives of pyridone, alkadiene, benzoquinone, pyridine, oxalylamino acid and proline analogs. However, all of these drugs suffer from the drawback of also inhibiting the normal, and required, synthesis of collagen as well as the detrimental synthesis that occurs during fibrosis.
One of the most important pathologies for which fibrosis is a contributing factor is cardiovascular disease. Cardiovascular disease is the leading cause of death in the Western world. In the United States it accounted for 930,000 deaths in 1990. There are an estimated 1.5 million heart attacks per year in the U.S. that result in more than 500,000 deaths annually.
Another fibrotic disease is proliferative vitreoretinopathy (PVR), which is characterized by the formation of a membrane in front and/or behind the retina, which is composed of ECM and cells. Some of the events thought to contribute to pathogenesis include migration of the retinal pigment epithelial (RPE) cells and retinal glial cells (Muller cells), and synthesis of extracellular molecules such as collagen. Pastor, J. C. (1998) Surv. Ophthalmol. 43:3. Extracellular matrix (ECM) components such as collagen bind to cells via integrins such as xcex12xcex21, and this interaction is likely to be integral to contraction. Schiro, J. A. et al. (1991) Cell 67:403; Gullberg, D. A. et al. (1990) Exp. Cell Res. 186:264. The typical PVR membrane is mainly composed of collagen I, II, and III, Jerdan, J. A. et al. (1989) Ophthalmology 96:801, and is found on the inner or outer surface of the retina, or along the posterior portion of the vitreous, Michels, R. G. et al. (1990) Retinal Detachment 1990:669.
Contraction of the epiretinal membrane results in tractional retinal detachment (TRD). Michels, R. G. et al. (1990) Retinal Detachment 1990:669; Pastor, J. C. (1998) Surv. Opthalmol. 43:3. Once the retina loses its functional contact with the underlying layer of retinal pigment epithelial (RPE) cells, it is irreversibly damaged due to apoptosis of the photoreceptors. Berglin, L. et al. (1997) Graefes Arch. Clin. Exp. Ophthalmol. 235:306; Cook, B. et al. (1995) Invest. Ophthalmol. Vis. Sci. 36:990. PVR occurs in up to 10% of patients undergoing surgery to reattach the retina. The Retina Society Terminology Committee (1983) Opthalmology 90:121. The prognosis for an individual afflicted by PVR is generally poor, and 20 to 40% of the patients lose their vision despite additional retinal reattachment surgeries. Michels, R. G. et al. (1990) Retinal Detachment 1990:669.
Growth factors such as transforming growth factor-xcex2 (TGF-xcex2), Connor, T. B. et al. (1989) J. Clin. Invest. 83:1661; Kon, C. H. et al. (1999) Invest. Ophthalmol. Vis. Sci. 40:705, and platelet-derived growth factor (PDGF), Robbins, S. G. et al. (1994) Invest. Ophthalmol. Vis. Sci. 35:3649; Campochiaro, P. A. et al. (1985) Arch. Ophthalmol. 103:576; Campochiaro, P. A. et al. (1994) J. Cell Sci. 107:2459; Cassidy, L. et al (1998) Br. J. Ophthalmol. 82:181; Garcia-Layana, A. et al. (1997) Curr. Eye Res. 16:556, are believed to play an important role in promoting the events which contribute to fibrotic diseases, such as PVR. Other growth factors, such as hepatocyte growth factor (HGF), Lashkari, K. et al. (1999) Invest. Ophthalmol. Vis. Sci. 40:149, basic fibroblast growth factor (bFGF), or interleukin-6 (IL-6), Kon, C. H. et al. (1999) Invest. Ophthalmol. Vis. Sci. 40:705; Cassidy, L. et al. (1998) Br. J. Ophthalmol. 82:181, have also been implicated.
PDGF is a potent mitogen for fibroblasts, and induces DNA synthesis, chemotaxis, and sometimes serves as a survival factor. Two PDGF gene have been identified, and they encode the PDGF-A and PDGF-B chain. Biologically active PDGF is either a homo- or heterodimer, therefore there are three kinds of combinations, PDGF-AA, -AB, and -BB. The receptor for PDGF is a homo- or heterodimer of the xcex1 and xcex2 subunits. The receptor subunits differ in their affinity for ligand, and hence the composition of receptor subunits is in part dependent on the isoform of PDGF. For instance, PDGF-AA only binds to xcex1xcex1 homodimer, -AB to xcex1xcex1 homo- or xcex1xcex2 heterodimer, and -BB binds to any subunit combination. In the studies described herein, we focus on the PDGF xcex1 receptor (xcex1PDGFR), which is a homodimer of the xcex1 subunits, and can be assembled by any of the three PDGF isoforms. PDGF dimerizes the xcex1PDGFR, leading to activation of the receptor""s tyrosine activity, which is encoded in the intracellular domain of the receptor. Activation of the receptor""s kinase as a prerequisite for subsequent signal relay and biological responses, such as cell migration, proliferation, synthesis and secretion of ECM, as well as contraction. The xcex1PDGFR can be activated by any of the PDGF isoforms (AA, AB, BB), including the newly discovered PDGF-CC isoform. Li, X. et al. (2000) Nat. Cell Biol. 2:302. Ligand binding activates the receptor, whereupon it becomes tyrosine phosphorylated and associates with a variety of SH2 domain-containing signaling enzymes. These include Src family kinases, the phosphotyrosine phosphatase SHP-2, phosphoinositide 3-kinase (PI3K), and phospholipase C-xcex31 (PLCxcex3). These signaling enzymes are required to mediate PDGF-dependent cellular responses, and different pathways seem to be involved in different biological reactions. For instance, PI3K is required to drive cells into S phase, whereas the combination of Src family kinase, PI3K, and PLCxcex3 are necessary for PDGF-dependent chemotaxis. Rosenkranz, S. et al. (1999) J. Biol. Chem. 274:28335.
Thus, it is desirable to have efficient agents for treating fibrotic diseases, such as PVR, as well as methods for identifying agents for treating fibrotic diseases.
In one embodiment, the invention provides methods and compositions for treating or preventing diseases that are associated with an abnormal PDGF level or PDGF-indcued biological response, such as cell proliferation, cell migration, extracellular matrix synthesis or secretion, and cell contraction. In a preferred embodiment, the invention provides methods and compositions for treating or preventing proliferative diseases, such as fibrotic diseases, e.g., proliferative vitreoretinopath (PVR), liver cirrhosis, pulmonary fibrosis, kidney fibrosis, scleroderma, keloids, hypertrophic scars, skin wound healing and atherosclerosis. The method preferably includes administering to a subject in need thereof, an amount of an agent sufficient to reduce a biological activity of PDGF. For example, the agent may inhibit receptor tyrosine kinases, e.g., platelet derived growth factor receptor (PDGFR). In an even more preferred embodiment, the agent inhibits at least part of the signal transduction from the xcex1PDGFR. For example, the agent inhibits activation of a Src family kinase, e.g., phosphoinositide 3-kinase (PI3K) and phospholipase C-xcex31 (PLCxcex3).
The agent for use in the methods of the invention can be a compound which inhibits receptor tyrosine kinases, e.g., PDGFRs. A preferred agent is a compound which prevents at least part of the signal transduction from such a receptor. The agent can be a small molecule, a peptide, or a nucleic acid. A preferred agent is a mutated form of a receptor tyrosine kinase, which acts, e.g., by competition with the wildtype receptor. Even more preferred agents are mutants of xcex1PDGFR or xcex2PDGFR, such as those further described herein. Other agents that can be used include ligands which compete with the naturally occurring ligand, e.g., with PDGF.
The agents can be administered together with a pharmaceutical carrier or excipient. In a preferred embodiment, the agent is administered locally to a subject in need thereof, e.g., in the eye in the case of proliferative vitreoretinopathy.
If the agent is a peptide or protein, e.g., a mutant of xcex1PDGFR, the agent can be administered to a subject as a nucleic acid encoding the peptide or protein. The nucleic acid can be administered as naked DNA, or it can be combined with an agent facilitating its delivery, e.g., liposomes. A preferred method of administering a nucleic acid is by administering a viral vector containing the nucleic acid. The viral vector can be, e.g., an adenovirus, an adenovirus-associated virus (AAV), a herpes virus, a papillomavirus, or a retrovirus. In a preferred embodiment, a viral vector encoding a truncated xcex1PDGFR is administered to a subject having or being likely to develop, a fibrotic disease.
In some embodiments, it may be desirable to target the agent or the vector encoding the agent to a specific tissue, e.g., retina. This can be acccomplished by various means, e.g., by using a viral vector that is specific for the desired target tissue. Alternatively, the viral vector or liposome or other carrier can be modified to express on its surface a molecule that will interact with a molecule on the surface of the target tissue.
The agents for use in the methods are also within the scope of the invention. Preferred agents include mutant xcex1PDGFRs and mutant xcex2PDGFRs and nucleic acids encoding such. For example, the invention provides polypeptides comprising an amino acid sequence having the general structure X-Y-Z, wherein Y consists of a portion of platelet derived growth factor-alpha receptor (PDGFxcex1R) consisting essentially of amino acids 1 to about 589; amino acids 21 to about 589; or amino acids 25 to about 589 of SEQ ID NO: 2; X and Z consist of at least one amino acid, wherein, if Z is more than one amino acid, Z does not have the amino acid sequence of human PDGFxcex1R located downstream of about amino acid 589. Y can also be amino acids 1 to about 561 of SEQ ID NO: 25 (beta PDGFR). In one embodiment, X and Z are absent. In another embodiment, the invention provides polypeptides consisting essentially of, comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 4, 6, 8, or 10 (PDGFRalpha mutants) or SEQ ID NO: 16, 18, 20, 22, or 24 (PDGFR beta mutants). These polypeptides may lack the signal peptide.
Other preferred agents of the invention are nucleic acids encoding a mutant PDGFR polypeptide, e.g., nucleic acids comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 3, 5, 7, 9, 15, 17, 19, 21 and 23. These nucleic acids are preferably operably linked to at least one transcriptional regulatory element, and may be part of a vector. The invention further provides pharmaceutical compositions including a nucleic acid or polypeptide of the invention, and methods for preparing such pharmaceutical compositions.
The invention further provides assays for identifying agents which can be used to treat fibrotic diseases and/or diseases associated with an abnormal contraction of cells. In a preferred embodiment, the assay comprises contacting cells, e.g., fibroblasts, with an agent and determining whether the cells contracted. In an even more preferred embodiment, the cells and the agent are suspended in a matrix, e.g., a collagen matrix. It has been shown herein that use of this in vitro assay correlates with in vivo tests of activity of compounds.